ABSTRACT Potassium (K+) channels are major determinants of cell excitability and play crucial roles in physiological processes. Specifically, large conductance and Ca2+-activated K+ (BK) channels, have the ability to couple intracellular Ca2+ to membrane potential variations, play major physiological roles ranging from vascular smooth muscle tone maintenance and regulation of circadian rhythms, to hearing, regulating neuronal firing, and neurotransmitter release. BK channel dysfunction has been associated with many pathophysiological conditions, so understanding Ca2+-gating can have major therapeutic consequences. The overall objective of this grant is to understand molecular mechanisms of Ca2+-gating in K+ channels (opening, closing, and inactivation) by employing functional, structural, and computational analysis on a model BK channel, MthK, a close prokaryotic homolog. Unlike BK channels, where voltage-dependent gating is interfering with Ca2+-gating thus preventing structural determination of specific conformations, MthK is devoid of voltage sensors and thus a perfect system for investigating Ca2+-dependent gating alone. In addition, electrophysiology experiments suggested that MthK, just like BK, lacks inactivation at the selectivity filter. This is intriguing, because BK and MthK share high sequence and structure similarity in the filter with other ?inactivating? K+ channels. However, in specific conditions of K+ and bilayer thickness, MthK does inactivate, raising the possibility that BK also inactivates under these conditions. This can have major physiological consequences, as knowing conditions that control activity will lead to new understanding of BK channels? role in different cell types and cellular locations. Our first aim is to determine the molecular mechanism of Ca2+-activation. We propose to determine the structures of apo and Ca2+ bound MthK, from channels reconstituted in lipid nanodiscs, using single- particle cryo-EM. Lipid composition will be adjusted to yield an open state. MD simulations will be employed to refine the structures in the lipid membrane, simulate K+ flux, and uncover possible activation pathways. Our second aim is to determine the lipid bilayer-dependent inactivation mechanism. We will systematically investigate MthK activation and inactivation kinetics in liposomes of varying lipid thickness made by varying lipid lengths. Preliminary stopped-flow functional data revealed that thinner bilayers promote MthK inactivation. Using single-particle cryo-EM, we will determine structures of MthK in nanodiscs of different lipid composition and associate functional states directly from functional assays. MD simulations will refine these structures, as well as observe how changing membrane thickness affects conformation and conduction. In our third aim, to understand the more subtle changes that lead to inactivation in MthK, we propose to use X-ray crystallography of pore-only MthK together with MD simulations to reveal the sequence of molecular changes involving selectivity filter, lower gate, ions and water, by imposing conditions that promote inactivation in functional assays. The accomplishment of these aims will provide a comprehensive picture of Ca2+-gating in K+ channels.